Potent and Selective Benzothiazole-Based Antimitotics with Improved Water Solubility: Design, Synthesis, and Evaluation as Novel Anticancer Agents

The design of colchicine site ligands on tubulin has proven to be a successful strategy to develop potent antiproliferative drugs against cancer cells. However, the structural requirements of the binding site endow the ligands with low aqueous solubility. In this work, the benzothiazole scaffold is used to design, synthesize, and evaluate a new family of colchicine site ligands exhibiting high water solubility. The compounds exerted antiproliferative activity against several human cancer cell lines, due to tubulin polymerization inhibition, showing high selectivity toward cancer cells in comparison with non-tumoral HEK-293 cells, as evidenced by MTT and LDH assays. The most potent derivatives, containing a pyridine moiety and ethylurea or formamide functionalities, displayed IC50 values in the nanomolar range even in the difficult-to-treat glioblastoma cells. Flow cytometry experiments on HeLa, MCF7, and U87MG cells showed that they arrest the cell cycle at the G2/M phases at an early time point (24 h), followed by apoptotic cell death 72 h after the treatment. Tubulin binding was confirmed by microtubule network disruption observed via confocal microscopy. Docking studies support favorable interaction of the synthesized ligands at the colchicine binding site. These results validate the proposed strategy to develop potent anticancer colchicine ligands with improved water solubility.


Introduction
Cancer is one of the main causes of death worldwide, responsible for approximately one of every six deaths and accounting for nearly ten million deaths in 2020 [1]. Cancer is defined as a group of diseases characterized by the uncontrolled division of abnormal cells beyond their usual boundaries as a consequence of failures of the systems of regulation of cell proliferation and homeostasis. Although cancer research in the past two decades has allowed us to better understand the cellular mechanisms involved in the development of the disease, and many new targets for cancer treatments were identified, the difficulties in achieving high anticancer potency and no side effects together with good pharmacokinetic profiles make the development of effective chemotherapy one of the biggest challenges of pharmaceutical sciences [2].
A considerable number of anticancer drug targets are cell cycle related since it is one of the sources of cell proliferation deregulation. The cell cycle consists of a series of Different from the taxanes and Vinca alkaloid sites in tubulin, the colchicine site, located at the interface between the α and β tubulin subunits, is appropriate for the design of ligands because it has synthetically accessible simple structures [11][12][13]. In recent years, the colchicine site ligands have gained attention since they behave not only as antiproliferative drugs with a high potency against cancer cells, but also as vascular disrupting agents [14]. According to structural studies, the colchicine site is composed of three connected sub-pockets, namely, B-A-C [15][16][17]. Few examples of ligands binding simultaneously to the three zones were reported, such as Lexibulin [18] and ABT-751 [19] ( Figure  1b). The most frequently reported colchicine ligands consist of two aromatic rings linked by different functional groups that bind to two of the colchicine zones, usually zones A-B, such as combretastatin A-4 (CA-4) and its derivatives [20] (Figure 1c), known as classical ligands, which were intensively studied due to their high antimitotic potency [21][22][23]. However, some concerns are involved in the clinical limitations of CA-4 derivatives such as their low water solubility and their susceptibility to phase II metabolic transformations by UDP-glycosyltransferases through the hydroxy groups, which is usually followed by the development of drug resistance [24]. Structural modifications of combretastatin Another approach to obtain more soluble and potent tubulin ligands is the design of molecules targeting the colchicine zones A-C since their amino acid residues are more polar compared with zones A-B [38]. The success of this strategy can be illustrated by the antimitotic agents nocodazole [39] and MI-181 ( Figure 1d) [40], a benzothiazole derivative.
MI-181 is a potent antitumor agent acting as an inhibitor of tubulin polymerization. It can inhibit the cell proliferation of HeLa cells with an IC 50 value of 17 nM. Even though it is more soluble than combretastatin A4 (5.6 µg/mL vs. 1 µg/mL for CA-4), its water solubility is still limited [41]. However, its interaction in the tubulin site was investigated, and its simple structure offers great opportunities to perform chemical modifications aimed to improve its physicochemical and pharmacological properties.
The main objective of this work is the design, synthesis, and biological evaluation of new colchicine site ligands with an improved aqueous solubility, together with a high binding affinity to tubulin, that trigger an antimitotic effect on cancer cells. With this aim, the olefin linker of combretastatins and MI-181 was successfully substituted by a methylamino bridge. As a combination of structural features of the benzothiazole ring of MI-181 and the p-methoxyphenyl moiety of Combretastatin A-4, we propose 6-methoxybenzothiazole as the B ring. We further explored the incorporation of the following different aromatic A rings: 2-, 3-, or 4-pyridine; benzene; 5-metyl-1H-imidazole; and naphthalene. Finally, substitutions on the methylamino group to obtain the corresponding formamide, acetamide, or ethylurea derivatives were undertaken ( Figure 2). Some of the newly synthesized compounds are more water-soluble than the references MI-181 and ABT-751. Their antiproliferative activity was studied in a selection of human tumor cell lines representative of the kinds of cancer that have the highest incidence worldwide, including cervix uterine, breast, colon, liver, prostate, and brain cancer. Embryonic kidney HEK-293 cells were used to study toxicity in healthy tissues. Multidrug resistance and the mechanism of action were investigated for the most potent compounds. Docking studies were performed for all the new molecules.

General Chemical Techniques
Reagents were used as purchased without further purification. Solvents (toluene, ethanol, EtOAc, CH2Cl2, MeOH) were dried and stored over molecular sieves. TLC was performed on precoated silica gel polyester plates (0.25 mm thickness) with a UV fluorescence indicator 254 (Polychrom SI F254). Chromatographic separations were performed on silica gel columns via flash (Kieselgel 40, 0.040-0.063; Merck, Rahway, NJ, USA) chromatography. 1 H NMR and 13 C NMR spectra were recorded in CDCl3, CD3OD, DMSO-d6, or acetone-d6 on a Bruker SY spectrometer at 400/100 MHz or on a Varian Mercury 400/100 MHz spectrometer. Chemical shifts (δ) are given in ppm downfield from tetramethylsilane, and coupling constants (J values) are given in Hz. IR spectra in KBr disks were run on a Nicolet Impact 410 Spectrophotometer. A hybrid QSTAR XL quadrupole/time of flight spectrometer was used for HRMS analyses. The aqueous solubility of the compounds was determined in a Helios Alfa Spectrophotometer from Thermo-Spectronic.

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A catalytic amount of p-toluenesulfonic acid (0.1 eq) was added to a solution of 6methoxybenzo[d]thiazol-2-amine (1 eq) and the aromatic aldehyde derivative (1 eq) in dry toluene (30 mL). A Dean-Stark system was used to remove the water, and the mixture was stirred for 16 h under reflux. Then, the solvent was evaporated under reduced pressure, and the residue was re-dissolved in absolute ethanol (20 mL). NaBH4 (1.5 eq) was added at 0 °C, and the mixture was stirred at room temperature for 2 h. Then, the solvent was removed under vacuum, and the residue was dissolved in CH2Cl2. The organic layer was washed with saturated NaCl water, dried over anhydrous Na2SO4, filtered, and evaporated under vacuum.

General Chemical Techniques
Reagents were used as purchased without further purification. Solvents (toluene, ethanol, EtOAc, CH 2 Cl 2 , MeOH) were dried and stored over molecular sieves. TLC was performed on precoated silica gel polyester plates (0.25 mm thickness) with a UV fluorescence indicator 254 (Polychrom SI F254). Chromatographic separations were performed on silica gel columns via flash (Kieselgel 40, 0.040-0.063; Merck, Rahway, NJ, USA) chromatography. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 , CD 3 OD, DMSOd 6 , or acetone-d 6 on a Bruker SY spectrometer at 400/100 MHz or on a Varian Mercury 400/100 MHz spectrometer. Chemical shifts (δ) are given in ppm downfield from tetramethylsilane, and coupling constants (J values) are given in Hz. IR spectra in KBr disks were run on a Nicolet Impact 410 Spectrophotometer. A hybrid QSTAR XL quadrupole/time of flight spectrometer was used for HRMS analyses. The aqueous solubility of the compounds was determined in a Helios Alfa Spectrophotometer from Thermo-Spectronic.

Chemical Synthesis and Characterization Method A. Reductive Amination (Compounds 1-6)
A catalytic amount of p-toluenesulfonic acid (0.1 eq) was added to a solution of 6methoxybenzo[d]thiazol-2-amine (1 eq) and the aromatic aldehyde derivative (1 eq) in dry toluene (30 mL). A Dean-Stark system was used to remove the water, and the mixture was stirred for 16 h under reflux. Then, the solvent was evaporated under reduced pressure, and the residue was re-dissolved in absolute ethanol (20 mL). NaBH 4 (1.5 eq) was added at 0 • C, and the mixture was stirred at room temperature for 2 h. Then, the solvent was removed under vacuum, and the residue was dissolved in CH 2 Cl 2 . The organic layer was washed with saturated NaCl water, dried over anhydrous Na 2 SO 4 , filtered, and evaporated under vacuum. Ethylisothiocyanate (2.5 eq) and triethylamine (2.5 eq) were added to a solution of the 6-methoxybenzo[d]thiazol-2-amine derivative 1, 2, 3, 4, 5, or 6 in dry CH 2 Cl 2 (12-13 mL). The solution was stirred overnight, at room temperature, under N 2 atmosphere. Then, the solvent was removed under vacuum, and the residue was dissolved in CH 2 Cl 2 . The organic layer was washed with saturated NaHCO 3 water solution and saturated NaCl water solution, dried over anhydrous Na 2 SO 4 , filtered, and evaporated under vacuum.

Determination of Aqueous Solubility
The aqueous solubility of compounds was measured in a Helios Alfa Spectrophotometer. Calibration lines at the maximum wavelength of absorbance were determined for each compound. Then, following the shake-flask method, a suspension of 5 mg of each compound in 500 µL in pH 7.0 buffer was stirred for 72 h at room temperature. The mixture was then filtrated over a 45 µm filter to remove the insoluble fraction. The concentration of the compound in the resulting saturated solution was determined via UV absorbance measurement and interpolation in the calibration line. The aqueous solubility values are given as the average of three independent measurements.

Calculation of Properties
The total polar surface area (TPSA) and the logarithm of the partition coefficient (Log P o/w ) were calculated using SwissADME software [42,43]. TPSA calculations follow a methodology based on a sum of fragment-based contributions [44]. The consensus Log P o/w , which is the arithmetic mean of the values predicted by the five proposed methods applied by Swiss-ADME, was used in this work.

Cell Growth Inhibition Assay
Cells were plated in 96-well plates (100 µL/well) at the following concentrations: 30,000 cells/mL (HepG2, U87 MG, T98G, C6, LNCaP, PC3, and HT-29 cells) or 15,000 cells/mL (HeLa and MCF-7 cells). Previous experiments were performed to choose the optimal cell concentration (70-80% confluence) and avoid the formation of cell aggregates. Cells were incubated in complete DMEM or RPMI 1640 medium (see above) at 37 • C and 5% CO 2 atmosphere for 24 h to allow for cell attachment to the plate. Then, every compound was added (10 µL/well) to a final concentration of 10 µM. Non-treated cells were used as negative controls. The antiproliferative activity of compounds was measured 72 h after the treatments using the MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), (MTT Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS at 5 mg/mL. Compounds showing antiproliferative effects at 10 µM were selected to determine their IC 50 value. For that purpose, compounds were used at different concentrations ranging from 10 −10 to 10 −2 M. Measurements were performed in triplicate, and each experiment was repeated three times. The IC 50 value was determined for every compound using Origin software (OriginLab, Washington, USA). To study the sensitivity to MDR efflux pumps of compounds, the IC 50 values were determined by following the same procedure but in the presence of verapamil (final concentration of 10 µM).

Tubulin Polymerization Inhibition
Microtubular protein was isolated from calf brain following the modified Shelanski method, based on temperature-dependent assembly/disassembly of tubulin [45]. The protein was stored at −80 • C, and its concentration was determined using the Bradford method before each use. Tubulin polymerization, which occurs when heating a buffered tubulin sample from 4 • C to 37 • C, is accompanied by an increase in the absorbance (at 450 nm) of the sample that was monitored by a Helios α spectrophotometer. The samples containing 1.5 mg/mL of microtubular protein at pH 6.7 buffer (0.1 M MES, 1 mM β-ME, 1 mM EGTA, 1 mM MgCl 2 , and 1.5 mM GTP) and the ligand at 10 µM (except for control samples) were incubated at 20 • C for 30 min to allow for the ligand to bind to the tubulin, and were subsequently cooled on ice for 10 min to make sure that polymerization did not take place at the initial time point. Then, the temperature was adjusted to 4 • C, and the temperature being shifted to 37 • C allowed for the assembly process of tubulin. The turbidity caused by tubulin polymerization was measured by the increased absorbance at 450 nm. The increase in absorbance obtained for the control sample (without the ligand), which we considered to be 100% tubulin polymerization (0% inhibition), compared with the increase in absorbance determined for the sample containing ligands yielded the degree of tubulin polymerization inhibition (TPI), which is expressed as a percentage value. For the compounds displaying TPI values over 50% at 10 µM, the IC 50 value for TPI was calculated. Measurements were performed in triplicate in two independent experiments using microtubular protein from different preparations.

Immunofluorescence Experiments
The 8 × 10 4 HeLa, MCF7, and U87 MG cells were seeded on 0.01% poly-L-lysine pre-coated square glass coverslips (22 mm 2 ) deposited on 6-well plates (1 coverslip/well) and incubated in complete DMEM medium (see above) at 37 • C and 5% CO 2 atmosphere for 24 h. Then, the culture medium was replaced by fresh complete DMEM, and cells were incubated in the presence or absence of selected compounds for 24 h. Untreated cells were used as negative controls. The concentration of the compounds was selected according to their IC 50 value for the antiproliferative activity in each cell line as follows: 50 nM if IC 50 is in the low nanomolar range (below 10 nM); 100 nM when IC 50 is in the two-digit nanomolar; 200 nM when IC 50 is near 100 nM, and 500 nM for compounds with IC 50 near 200 nM. In that way, compounds with similar antiproliferative potencies were tested at the same concentrations. HeLa cells were treated with 200 nM of compounds 8 and 14 and 500 nM of compound 9; MCF7 cells were treated with 100 nM of compounds 8 and 14 and 500 nM of compound 9; and U87 MG cells were treated with 50 nM of compound 8 and 100 nM of compounds 9 and 14. After incubation, medium was removed and coverslips were washed three times with PBS, fixed in 4% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 90 s at 4 • C, and blocked with 10% BSA in PBS for 30 min. After four washes with PBS, the coverslips were incubated for 1 h with anti-α-tubulin mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), and diluted 1:200 in PBS containing 3% BSA. After four washes wish PBS, coverslips were incubated, in darkness, with fluorescent secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Invitrogen, Eugenen, OR, USA), and diluted 1:400 in PBS containing 1% BSA for 1.5 h. After four washes with PBS, a drop of ProLong™ Gold Antifade Mountant containing DAPI (ThermoFisher, Waltham, MA, USA) was added for cell nuclei staining. Samples were analyzed via confocal microscopy using a LEICA SP5 microscope DMI-6000V model coupled to a LEICA LAS AF software computer.

Cell Cycle Analysis
An amount of 8 × 10 4 cells/mL (HeLa, MCF7, or U87 MG cells) were seeded in 6-well plates (2 mL/well) and incubated in complete DMEM medium (see above) at 37 • C and 5% CO 2 atmosphere for 24 h. Then, the medium was replaced with fresh complete DMEM in the presence or absence of the selected compounds (8, 9, and 14). The concentration of the compounds was selected according to their IC 50

Apoptotic Cell Death Quantification
Annexin V-FITC/PI apoptosis detection kit (Immunostep, Salamanca, Spain) was used to quantify HeLa, MCF7, or U87 MG cells by following the manufacturer's guidelines. An amount of 5 × 10 4 cells/mL were seeded in 12-well plates (1 mL/well) and incubated in complete DMEM medium (see above) at 37 • C and 5% CO 2 atmosphere for 24 h. Then, the medium was replaced with fresh complete DMEM in the presence or absence of the selected compounds (8, 9, and 14). The concentration of the compounds was selected according to their IC 50 value for the antiproliferative activity in each cell line (see above, Section 2.4.4). HeLa cells were treated with 200 nM of compounds 8 and 14 and 500 nM of compound 9; MCF7 cells were treated with 100 nM of compounds 8 and 14 and 500 nM of compound 9; and U87 MG cells were treated with 50 nM of compound 8 and 100 nM of compounds 9 and 14. Untreated cells were used as negative controls. After 72 h of incubation, cells were collected, centrifugated, resuspended in the Annexin V binding buffer, and stained with Annexin V-FITC/PI. Cells were then incubated in darkness for 15 min at room temperature. Samples were analyzed using BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences), and acquired data were analyzed using BD Accuri™ C6 Software (version 1.0.264.21).

Lactate Dehydrogenase Assay
CytoTox96 ® Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA) was used to measure the release of lactate dehydrogenase (LDH) by following the previously described method in [46]. Briefly, U87 MG or HEK-293 cells were incubated for 72 h with compounds 8, 9, or 14 at concentrations ranging from 10 −9 to 10 −6 M for U87 MG cells, and from 10 −4 to 10 −3 M for HEK-293 cells. After treatment, the supernatants were collected, and the intact attached cells were lysed using 0.1% (w/v) Triton X-100 in (0.9%) NaCl solution. Both the LDH released to culture media, as well as the LDH content within the cells, were determined spectrophotometrically at 490 nm on a 96-well plate reader (Infinite 200, Tecan, Salzburg, Austria) by following the manufacturer's instructions. LDH release was defined by the ratio of LDH released/total LDH present in the cells, with the total LDH being 100%. All the samples were run in quadruplicate.

Computational Studies
Structural preferences for the unsubstituted scaffolds (N-benzylbenzothiazole-2-amine, N-benzyl-N-(benzothiazole-2-yl)formamide, N-benzyl-N-(benzothiazole-2-yl)acetamide, and N-benzyl-N-(benzothiazole-2-yl)-N'-ethylurea) were calculated by means of RB3LYP DFT calculations at the 6-31G(D) level with Spartan 08 software package. The structures of every possible isomer for each ligand were built and subjected to conformational searches at the molecular mechanics (MMFF force field) level. The retrieved conformations were then minimized with RB3LYP DFT calculations at the 6-31G(D) level, and the lowest energy configurations amongst all the possible outcomes were selected as the most stable forms for every compound.
We carried out ensemble docking studies as previously described, with the tubulin flexibility accounted for by the use of different complexes that sample the protein conformational space [16,47]. Briefly, we docked the compounds into the colchicine sites of 65 different tubulin structures from complexes of tubulin with colchicine site ligands deposited in the pdb, and 5 representative structures from a previous molecular dynamics simulation run on a tubulin-podophyllotoxin complex. We performed parallel docking studies using AutoDock 4.2 with the Lamarckian genetic algorithm (LGA) 100−300 times for a maximum of 2.5 million energy evaluations, 150 individuals, and a maximum of 27,000 generations, and used PLANTS with default settings and 10 runs per ligand. For ligands with amide bonds and urea groups, we used all the possible configurations in the docking runs, and selected the best scored one as the docking results for every ligand. We converted the scores of the different programs into Z-scores to allow for the comparison of the different scoring scales. We selected the common poses for the two programs with the best consensus Z-scores as the docking results. For every pose, we automatically assigned them to the colchicine subzones using in-house KNIME pipelines [48]. The RMSD between every pose and model scaffolds with no substituents and with the colchicine binding site ligands representative of the binders occupying different subzones were calculated using LigRMSD [49]. Docked poses were analyzed using Chimera [50], Marvin [51], OpenEye [52], and JADOPPT [53].

Chemical Synthesis
The N-arylmethylbenzothiazole amines 1-6 were prepared via reductive amination in two steps. First, the imines were synthesized under acid catalysis (p-toluenesulphonic acid, pTsOH), and then sodium borohydride was used for the reduction in the intermediate imines. The structural modifications on the linker connecting the aromatic rings were accomplished via acylation of the secondary amino group of the benzothiazole derivatives, leading to the following three families of compounds: urea (7-12), formamide (13)(14)(15)(16)(17)(18), and acetamide (19-24) derivatives (Scheme 1). We carried out ensemble docking studies as previously described, with the tubulin flexibility accounted for by the use of different complexes that sample the protein conformational space [16,47]. Briefly, we docked the compounds into the colchicine sites of 65 different tubulin structures from complexes of tubulin with colchicine site ligands deposited in the pdb, and 5 representative structures from a previous molecular dynamics simulation run on a tubulin-podophyllotoxin complex. We performed parallel docking studies using AutoDock 4.2 with the Lamarckian genetic algorithm (LGA) 100−300 times for a maximum of 2.5 million energy evaluations, 150 individuals, and a maximum of 27,000 generations, and used PLANTS with default settings and 10 runs per ligand. For ligands with amide bonds and urea groups, we used all the possible configurations in the docking runs, and selected the best scored one as the docking results for every ligand. We converted the scores of the different programs into Z-scores to allow for the comparison of the different scoring scales. We selected the common poses for the two programs with the best consensus Z-scores as the docking results. For every pose, we automatically assigned them to the colchicine subzones using in-house KNIME pipelines [48]. The RMSD between every pose and model scaffolds with no substituents and with the colchicine binding site ligands representative of the binders occupying different subzones were calculated using LigRMSD [49]. Docked poses were analyzed using Chimera [50], Marvin [51], OpenEye [52], and JADOPPT [53].

Chemical Synthesis
The N-arylmethylbenzothiazole amines 1-6 were prepared via reductive amination in two steps. First, the imines were synthesized under acid catalysis (p-toluenesulphonic acid, pTsOH), and then sodium borohydride was used for the reduction in the intermediate imines. The structural modifications on the linker connecting the aromatic rings were accomplished via acylation of the secondary amino group of the benzothiazole derivatives, leading to the following three families of compounds: urea (7-12), formamide (13)(14)(15)(16)(17)(18), and acetamide (19-24) derivatives (Scheme 1). The transformations of the secondary amino groups into ethylurea to obtain compounds 7-12 were performed by the reaction between derivatives 1-6 and ethylisocyanate under basic conditions. The formamide derivatives 13-18 were obtained via the amide coupling reaction between the secondary amines of compounds 1-6 and formic acid, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-hidroxybenzotriazole (OHBt), and 4-dymethylaminopyridine (DMAP). The acetylation of the amines 1-6 with acetic anhydride afforded the acetamide derivatives 19-24 in a reaction catalyzed by DMAP. All the products were obtained in good or excellent yields. In the NMR spectra of the formamide derivatives, it was possible to distinguish the signals corresponding to both the cis and trans isomers. This can be explained by the energetic barrier to interconversion in the formamides that allows for the observation of both isomers in the NMR chemical shift timescale [54].

Aqueous Solubility and Calculated Properties
One of the main drawbacks of the classical colchicine ligands is their low aqueous solubility. The high hydrophobicity of zones A and B in the colchicine site in the tubulin implies that the ligands with a high binding affinity, such as combretastatin A-4, exhibit non-drug-like physicochemical properties as a consequence of their hydrophobic structure. The development of moleculesadapted to interact with the more polar zone C is usually related to the design of polar ligands, such as the MI181, which binds zones A-C, or the sulfonamide ABT-751, which targets zones B-A-C. One of the goals of the design of 6methoxybenzothiazol-2-amine derivatives prepared in this work is the substitution of the olefin bridge of combretastatins and MI-181 by amino, amido, or urea groups to improve the aqueous solubility. The thermodynamic water solubility of the synthesized compounds, in the phosphate buffer at pH 7.0, was determined using the shaking flask methodology. Combretastatin A4, MI-181, and ABT-751 were used as references, and the results are summarized in Table 1. The topological polar surface area (TPSA) and the logarithm of partition coefficient (Log P) were computationally calculated as descriptors related to water solubility, toxicity, and the ability of compounds to cross biological barriers (Table 1) [55].
The nature of the second aromatic ring of the benzothiazole derivatives was the main factor affecting the aqueous solubility. The presence of the naphthalene ring imposed low water solubilities below 9 µg/mL in all cases (e.g., compounds 6, 12, 18, and 24), similar to the less soluble reference compounds MI-181 and CA-4. The substitution of the large hydrophobic naphthalene ring by a phenyl moiety increased the solubility by about two-fold (e.g., compare compound 5 with 6; 11 with 12; 17 with 18; and 23 with 24) with water solubilities between 5 and 16 µg/mL for the phenyl derivatives. The replacement of the phenyl ring by the pyridine greatly increased the water solubility, ranging from 6-to 11-fold (e.g., compare compound 5 with 2; 11 with 9; 17 with 14; and 23 with 20), yielding values of 48-57 µg/mL for the aminomethyl-pyridine derivatives (1-3) and about 90-100 µg/mL for the amido-and urea-pyridine derivatives.
The position of the nitrogen of the 2-, 3-, or 4-pyridyl moieties did not make significant differences in the solubility values (e.g., compare compounds 13, 14, and 15; or 7, 8, and  9). The best solubility results were obtained with a 5-methyl-1H-imidazole moiety, with improvements between 1.5-and 2-fold (e.g., compare compound 3 with 4, and 22 with 19). The substitution on the amino group of benzothiazole derivatives was the second factor affecting the water solubility. Introducing an ethylurea group caused an increase in the water solubility of about 2-fold in comparison with the amino series. For the pyridine derivatives, the water solubility improved from around 50 µg/mL in the amino series to near 100 µg/mL in the urea family (e.g., compare compound 7 with 1; 8 with 2; or 9 with 3). Similar trends were obtained for the substitutions of the amino groups by formamide (e.g., compare compound 13 with 1; 14 with 2; and 15 with 3, showing improvements from 1.6-to 1.8-fold). The behavior of the acetamide derivatives was similar to their formamide analogs (e.g., compare compound 13 with 19; 15 with 21; and 16 with 22). Therefore, the introduction of the ethylurea, formyl, or acetyl groups was favorable in terms of water solubility, which is probably explained by the higher topological polar surface area (TPSA) of the substituted derivatives with respect to the non-substituted ones (e.g., compare TPSA values of 1, 7, 13, and 19), as well as by the greater possibilities of the substituted derivatives to form hydrogen bonds with water. This last feature is the most relevant in the case of the urea derivatives having the highest solubility values (e.g., compare compound 7 with 13 or 19; 8 with 14 or 20; and 9 with 15 or 21). Anywhere, all the synthesized compounds were more soluble than the references MI-181 and CA-4, and those with pyridine or imidazole rings showed higher solubilities than ABT-751. By comparing the structure of MI-181 with the 3-pyridyl derivatives (compounds 2, 8, 14, and 20), we can conclude that replacing the olefin with an amino, formamido, acetamido, or ethylurea group, together with the substitution of two methyl groups in positions 5 and 6 by a methoxy group in position 6 of the benzothiazole, results in large water solubility improvements. indexes between three and six for the tumor cells. These results suggest that the new benzothiazole derivatives have a significant safety profile for their use as antitumor agents.

Sensitivity to MDR Efflux Pumps
The compounds exhibited higher IC50 values against the HT-29 cells compared with the rest of the cell lines, which agrees with the previously reported data for the colchicine site ligands. The low sensitivity of the HT-29 cells to the tubulin polymerization inhibitors were related to autophagy [59], activity blockage by UDP-glucuronidation [60], or multidrug resistance mechanisms (MDR efflux pump-like P glycoprotein, P-gp) [61]. The compounds lack functional groups that are compatible with glucuronidation reactions. Since an acquired resistance to antitumor drugs is one of the more important limitations of chemotherapy, we studied the possibility that these compounds are substrates of MDR efflux pumps. For that purpose, the IC50 values of the benzothiazole derivatives against the HT-29 cells pre-treated with verapamil, a P-glycoprotein 1 and multidrug resistance protein 1 inhibitor (MDR1), at 10 µM, which is a concentration that does not affect cell proliferation, were compared with the values in absence of the inhibitor. If a compound is susceptible to MDR efflux, its IC50 values will be significatively lower in the presence of verapamil. No differences were observed between the two experiments with or without verapamil (Table 3), as well as the reference compound ABT-751, thus suggesting that they are not MDR substrates. The calculated TPSA values are consistent with the measured solubility since higher TPSA values were obtained for the more soluble molecules. The TPSA correlates well with the passive molecular transport through membranes, and it is also a good predictor of non-specific toxicity, since below the threshold of 75 Å 2 , the molecules are suspected to display unspecific cytotoxicity [55]. For all the pyridine and imidazole derivatives, the calculated TPSAs were over 75 Å 2 , with values ranging from 91 to 111 Å 2 for the latter; 83-95 Å 2 for the pyridine derivatives with urea, formamide, and acetamide groups; and 75 Å 2 for the rest of the pyridine derivatives (compounds 1, 2, and 3). The phenyl and naphthyl derivatives not only showed the lowest water solubility, but also unfavorable TPSA values below 70 Å 2 .
The partition coefficient between n-octanol and water (Log P o/w ) is the classical descriptor for lipophilicity. A good cell permeability is associated with LogP o/w values between two and three [55]. The calculations predicted LogP o/w values within that range for most of the soluble compounds. The only exception is compound 7, with a higher LogP o/w than its analogs, which is consistent with the possible formation of an intramolecular hydrogen bond between the endocyclic nitrogen of pyridine and the urea group.

Antiproliferative Activity
The MTT method was used to measure the effect of the synthesized compounds 1-24 on the in vitro cell viability 72 h after drug exposure. The compounds were tested against a panel of ten different cell lines. Eight of them were human tumor cell lines representative of some of the cancers with the highest incidence worldwide, including HeLa cervix epithelioid carcinoma, MCF7 breast adenocarcinoma, U87MG glioblastoma, T98G temozolomide resistant glioblastoma, HepG2 hepatocellular carcinoma, HT-29 colon adenocarcinoma, and LNCaP and PC-3 prostate cancer. LNCaP is a hormone-sensitive cell line and represents an early stage of prostate cancer, whereas PC-3 is more resistant to chemotherapy and is a model for hormone-refractory prostate cancer. The compounds were also tested against the GL261 mice glioblastoma cell line that is usually studied previously to glioblastoma xenograft experiments in mice, and against the non-tumorigenic HEK-293 cell line from human embryonic kidney tissue to evaluate the selectivity of compounds between healthy cells and tumor cells. The compounds inhibiting cell proliferation by at least 40%, with respect to the negative control at 10 µM, were evaluated at concentrations in the range between 0.1 nM and 20 µM, and the half-maximal inhibitory concentrations (IC 50 ) were calculated (Table 2). ABT-751, docetaxel (DTX), and temozolomide (TMZ) were used for comparative purposes since the first one is an example of an antimitotic agent binding at the colchicine site with moderately good water solubility, DTX is a tubulin-binding agent in clinical use, and TMZ is the only approved treatment for glioblastoma. The results are summarized in Table 2.
With regard to the effect of the other aromatic rings of the benzothiazole derivatives on antiproliferative activity, the pyridyl derivatives were the best-performing compounds within each series. The phenyl analogs were less potent than the equivalent pyridines, with most of them showing IC 50 values in the low µM range. The imidazoles were all inactive, as well as the naphthalenes, with the only exception being the unsubstituted naphthyl methylamine 6, with antiproliferative values in the low µM range.
The pyridines exerted antiproliferative activities in all tumor cell lines with IC 50 values ranging from micromolar to low nanomolar depending on the substituent on the amino group and the cell line. The pyridyl derivatives with an unsubstituted amino group (compounds 1, 2, and 3) showed potencies in the one-digit micromolar range, circa 1 µM for HeLa, HepG2, MCF7, and U87MG, and between 2 and 3 µM for the rest of the tumor cell lines. Some differences were found for the different isomers of the pyridine analogs since the 3-pyridyl derivatives showed, in general, a lower IC 50 than the 4-pyridyl isomers, which, in turn, showed higher potencies than the 2-pyridyl derivatives (e.g., compare  compound 1, 2, and 3; 7, 8, and 9; 13, 14, and 15; or 19, 20, and 21). the HT-29 cells pre-treated with verapamil, a P-glycoprotein 1 and multidrug resistance protein 1 inhibitor (MDR1), at 10 µM, which is a concentration that does not affect cell proliferation, were compared with the values in absence of the inhibitor. If a compound is susceptible to MDR efflux, its IC50 values will be significatively lower in the presence of verapamil. No differences were observed between the two experiments with or without verapamil (Table 3), as well as the reference compound ABT-751, thus suggesting that they are not MDR substrates. For the pyridyl derivatives, the substitutions on the amino group greatly improved the antiproliferative potencies, particularly in the ethylurea (7, 8, and 9) and formamide series (13, 14, and 15), whereas a smaller improvement was observed in the acetylated analogs (19, 20, and 21). The functionalization with the ethylurea group decreased the IC 50 values from micromolar to submicromolar or nanomolar in most cell lines (compare 1, 2, and 3 with 7, 8, and 9, respectively), reaching higher potencies than the reference ABT-751 in all the cell lines. In the case of the 3-pyridyl derivatives, the changes were higher when moving from the amino (2) to the urea (8) derivative, resulting in double-digit nanomolar IC 50 values for compound 8, ranging from 25 to 94 nM in HeLa, HepG2, MCF7, T98G, GL261, and LNCaP, and rising to submicromolar values only for the more resistant cell lines PC-3 (161 nM) and HT-29 (208 nM). Interestingly, compound 8 was especially potent to inhibit cell proliferation in the glioblastoma cell lines, with an IC 50 value of 9 nM in U87MG. For the less sensitive glioblastoma cell line T98G, the IC 50 value was 52 nM, and 25 nM for GL261. These results are especially relevant, considering that the IC 50 of the only approved drug for the treatment of glioblastoma, TMZ, is over 100 µM in these glioblastoma cell lines. Compound 8 also showed lower IC 50 values than DTX against the U87 MG, T98G, GL261, and HT-29 cells. The ethylurea-pyridyl derivatives in compounds 7 and 9 also displayed lower IC 50 values than their non-substituted counterparts in compounds 1 and 3, respectively, showing between 6-and 20-fold decreases in the IC 50 values with potencies lower or similar to ABT-751. Their behavior was similar to that observed for compound 8 in the sense that higher potencies were obtained for the glioblastoma cell lines (double-digit nanomolar range in U87MG and GL261) compared with the rest of the cell lines (IC 50 values between 0.1 and 0.3 µM). However, in all the cell lines, compound 8 performed better than its analogues 7 and 9. The higher sensitivity of the glioblastoma cell lines is also remarkable when compared to the HeLa cells, which are usually highly sensitive to microtubule inhibitors [56]. These results suggest that the glioblastoma cells have a special sensitivity to this class of microtubule drugs. Besides the high sensitivity of the glioblastoma cell lines, the breast cancer cell line MCF7 also shows a higher sensitivity to these compounds, which is in agreement with the previous results for the colchicine site tubulin inhibitors [57,58].
The amine formylation also increased the antiproliferative potencies (compare com pounds 13, 14, and 15 with 1, 2, and 3, respectively), shifting the IC 50 values from the micromolar range for the unsubstituted compounds 1-3 to at least the submicromolar range for the formamide derivatives in compounds 13-15, thus performing better than or similar to the reference ABT-751. As observed in the ethylurea series, the 3-pyridyl isomer in compound 14 was also the most potent in all the cell lines, demonstrating a particularly strong effect not only in the glioblastoma cell lines, reaching IC 50 between 37 and 75 nM, but also in the MCF7 cells (15 nM The amine acetylation enhanced the IC 50 values with respect to the non-substituted analogs (e.g., compare compounds 1, 2, and 3 with 19, 20, and 21, respectively), but the effect was less pronounced than in the ethylurea or formamide series, since the improvements did not change the order or magnitude of the IC 50 values in most cases. The 3-pyridyl derivative of the acetamide series (compound 20) was again the most potent compound of the family, reaching submicromolar IC 50 values against the HepG2, MCF7, U87MG, T98G, and GL261 cells. The superiority of the 3-pyridyl isomers (when the amino group is substituted) in the glioblastoma cells was also maintained in this family. The 2-and 4-pyridyl analogs (compounds 19 and 21) led to little improvements with respect to their non-substituted counterparts (compounds 1 and 3, respectively), with IC 50 values in the low micromolar range.
Regarding the nature of the linker connecting the aromatic rings, the phenyl and pyridyl derivatives behave differently from the naphthalenes and imidazoles. For the first group, the amine acylation always results in potency improvements, while for the second one, acylation is detrimental. This suggests a size, shape, and probably polarity origin for the observed different behavior. For the six-membered rings, a higher effect is observed for the pyridines, which suggests the implication of a favorable polar interaction of the target with the azine nitrogen. The maximal effect within the pyridines is obtained for the 3-pyridyl isomers, with a geometry dependency that is also consistent with a polar directional interaction with the target that is optimal for the nitrogen in that position. The lower improvement observed for the 2-and 3-pyridyl isomers suggest a less optimal disposition for the directional bond, while the lower enhancement observed for the phenyl analogs is also consistent with the loss of such a favorable interaction.
All the compounds were also tested against the non-tumorigenic HEK-293 cell line, and all of them showed lower potencies than when they were tested against the tumor cell lines. Most of the compounds displayed no cytotoxic effect below 10 µM (compounds 1-6,  10-12, 16-18, 19, and 21-24). Interestingly, the most potent antiproliferative compounds against the tumor cells (the pyridyl derivatives in compounds 7-15, 13-15, and 20) showed a low cytotoxicity toward the HEK-293 cells, with IC 50 values between 8 and 10 µM and selectivity indexes between 25 and 100 for most of the cell lines, displaying selectivity indexes near 900 and 500 for the best-performing compounds (compound 8 against U87MG and compound 14 against MCF7, respectively). The selectivity indexes are much better than those for the reference compound ABT-751 and DTX, which had selectivity indexes between three and six for the tumor cells. These results suggest that the new benzothiazole derivatives have a significant safety profile for their use as antitumor agents.

Sensitivity to MDR Efflux Pumps
The compounds exhibited higher IC 50 values against the HT-29 cells compared with the rest of the cell lines, which agrees with the previously reported data for the colchicine site ligands. The low sensitivity of the HT-29 cells to the tubulin polymerization inhibitors were related to autophagy [59], activity blockage by UDP-glucuronidation [60], or multidrug resistance mechanisms (MDR efflux pump-like P glycoprotein, P-gp) [61]. The compounds lack functional groups that are compatible with glucuronidation reactions. Since an acquired resistance to antitumor drugs is one of the more important limitations of chemotherapy, we studied the possibility that these compounds are substrates of MDR efflux pumps. For that purpose, the IC 50 values of the benzothiazole derivatives against the HT-29 cells pre-treated with verapamil, a P-glycoprotein 1 and multidrug resistance protein 1 inhibitor (MDR1), at 10 µM, which is a concentration that does not affect cell proliferation, were compared with the values in absence of the inhibitor. If a compound is susceptible to MDR efflux, its IC 50 values will be significatively lower in the presence of verapamil. No differences were observed between the two experiments with or without verapamil (Table 3), as well as the reference compound ABT-751, thus suggesting that they are not MDR substrates.  6, 10-12, 16-18, 19, and 21-24). Interestingly, the most potent antiproliferative compounds against the tumor cells (the pyridyl derivatives in compounds 7-15, 13-15, and 20) showed a low cytotoxicity toward the HEK-293 cells, with IC50 values between 8 and 10 µM and selectivity indexes between 25 and 100 for most of the cell lines, displaying selectivity indexes near 900 and 500 for the best-performing compounds (compound 8 against U87MG and compound 14 against MCF7, respectively). The selectivity indexes are much better than those for the reference compound ABT-751 and DTX, which had selectivity indexes between three and six for the tumor cells. These results suggest that the new benzothiazole derivatives have a significant safety profile for their use as antitumor agents.

Sensitivity to MDR Efflux Pumps
The compounds exhibited higher IC50 values against the HT-29 cells compared with the rest of the cell lines, which agrees with the previously reported data for the colchicine site ligands. The low sensitivity of the HT-29 cells to the tubulin polymerization inhibitors were related to autophagy [59], activity blockage by UDP-glucuronidation [60], or multidrug resistance mechanisms (MDR efflux pump-like P glycoprotein, P-gp) [61]. The compounds lack functional groups that are compatible with glucuronidation reactions. Since an acquired resistance to antitumor drugs is one of the more important limitations of chemotherapy, we studied the possibility that these compounds are substrates of MDR efflux pumps. For that purpose, the IC50 values of the benzothiazole derivatives against the HT-29 cells pre-treated with verapamil, a P-glycoprotein 1 and multidrug resistance protein 1 inhibitor (MDR1), at 10 µM, which is a concentration that does not affect cell proliferation, were compared with the values in absence of the inhibitor. If a compound is susceptible to MDR efflux, its IC50 values will be significatively lower in the presence of verapamil. No differences were observed between the two experiments with or without verapamil (Table 3), as well as the reference compound ABT-751, thus suggesting that they are not MDR substrates.

Tubulin Polymerization Inhibition (TPI)
To determine whether the tubulin polymerization inhibition (TPI) is the mechanism of action responsible for the antiproliferative effect of the active compounds, their effect on the in vitro assembly of bovine brain tubulin was studied. The degree of tubulin polymerization in the presence or absence (negative control) of the compounds was determined via turbidimetry. All the compounds were assayed at a concentration of 10 µM, and for those inhibiting tubulin polymerization by more than 50% compared to the untreated control, the IC 50 values were determined.
The TPI and the antiproliferative activity were strongly correlated, since compounds with TPI IC 50 values lower than 5 µM showed IC 50 values in the submicromolar range, and the non-cytotoxic compounds were not able to inhibit tubulin polymerization at 10 µM (Table 4). This is in agreement with tubulin inhibition being the mechanism of the antiproliferative effect. Additionally, this lack of TPI activity for the non-antiproliferative compounds is due to a lack of activity on tubulin and not to a reduced uptake, which is in good agreement with the antiproliferative and verapamil cotreatment experiments. The pyridine derivatives behaved as the most potent inhibitors, following the same trends as in the antiproliferative activity. The pyridine derivatives with no substituents on the amino group (1, 2, and 3), showing micromolar antiproliferative activities, inhibited tubulin polymerization below 50% at 10 µM, whereas the pyridine derivatives with ethylurea (7, 8, and 9) or formamide functionalities (13, 14, and 15), whose antiproliferative IC 50 values where submicromolar or nanomolar, exhibited TPI IC 50 values between 2.9 and 5.0 µM, which is similar to or better than the reference compounds ABT-751 (4.4 µM), MI-181 (4.6 µM), and CA4 (2.8 µM). The acetylation of the bridge amino group did not greatly improve the TPI (e.g., compare 19, 20, and 21 with 1, 2, and 3, respectively), nor did it decrease the antiproliferative IC 50 by orders of magnitude. The best-performing ligands as tubulin polymerization inhibitors were again the most cytotoxic, namely, the 3-pyridyl derivatives in all series (compounds 2, 8, 14, and 20), and the most potent of them (compound 8) showed a TPI IC 50 value below that of the ABT-751 (2.9 vs. 4.4 µM). In the acetyl series, the 3-pyridyl derivative (20) was the only ligand with a TPI IC 50 value below 10 µM, and it was also the only compound in that series with submicromolar antiproliferative activity. This correlation strongly suggests that tubulin inhibition is behind the observed antiproliferative effects. The differences in the TPI values for the pyridine isomers also parallel the observed differences in the antiproliferative potency, which is in good agreement with the ranking order due to different interactions with the target and is consistent with the proposed directional polar interaction of the pyridine nitrogen with tubulin. Again, the potency drops in the TPI for the phenyl analogs; the putative lack of this interaction further supports this argument.
The naphthyl and imidazolyl derivatives (4, 6, 10, 12, 16, 18, 22, and 24), the less potent cytotoxic compounds, were not able to inhibit tubulin polymerization at 10 µM regardless of the substituents on the amino group, thus confirming that the replacement of pyridine by 5-methyl-imidazole or naphthalene hinders the interaction with tubulin, probably because an imidazole ring is too small and/or too polar to fit in the tubulin pocket, whereas the naphthalene moiety is too hydrophobic and/or too large. Even though compound 6 showed antiproliferative activity in the micromolar range in some cell lines, it failed to inhibit tubulin polymerization at 10 µM. The differences in the order of magnitude between the two experiments can be explained by the fact that the observation of the antiproliferative effect only needs the alteration of the tubulin polymerization dynamics at a low drug concentration, whereas the polymer mass changes at high protein and compound (micromolar) concentrations, which are required in TPI assays.

Effects on Cellular Microtubules
To confirm that the antiproliferative activity of the compounds in tumor cells is caused by the interference with tubulin polymerization, the effects of the representative compounds on cellular microtubules were investigated. For that purpose, we selected three of the most potent pyridyl derivatives with a substituent on the amino group, which are the ethylurea derivatives 8 (3-pyridyl derivative) and 9 (4-pyridyl derivative), and the 3-pyridyl formamide 14. We studied the effects of the three compounds on the microtubule networks of the HeLa, MCF7, and U87MG cells 24 h after the treatment via immunofluorescence confocal microscopy, labeling α-tubulin and the nuclei in comparison with the untreated cells ( Figure 3).
The naphthyl and imidazolyl derivatives (4, 6, 10, 12, 16, 18, 22, and 24), the less potent cytotoxic compounds, were not able to inhibit tubulin polymerization at 10 µM regardless of the substituents on the amino group, thus confirming that the replacement of pyridine by 5-methyl-imidazole or naphthalene hinders the interaction with tubulin, probably because an imidazole ring is too small and/or too polar to fit in the tubulin pocket, whereas the naphthalene moiety is too hydrophobic and/or too large. Even though compound 6 showed antiproliferative activity in the micromolar range in some cell lines, it failed to inhibit tubulin polymerization at 10 µM. The differences in the order of magnitude between the two experiments can be explained by the fact that the observation of the antiproliferative effect only needs the alteration of the tubulin polymerization dynamics at a low drug concentration, whereas the polymer mass changes at high protein and compound (micromolar) concentrations, which are required in TPI assays.

Effects on Cellular Microtubules
To confirm that the antiproliferative activity of the compounds in tumor cells is caused by the interference with tubulin polymerization, the effects of the representative compounds on cellular microtubules were investigated. For that purpose, we selected three of the most potent pyridyl derivatives with a substituent on the amino group, which are the ethylurea derivatives 8 (3-pyridyl derivative) and 9 (4-pyridyl derivative), and the 3-pyridyl formamide 14. We studied the effects of the three compounds on the microtubule networks of the HeLa, MCF7, and U87MG cells 24 h after the treatment via immunofluorescence confocal microscopy, labeling α-tubulin and the nuclei in comparison with the untreated cells ( Figure 3).  In the negative controls, for every kind of tumor cell, a characteristic morphology with hairy microtubule filaments, in green, covering all the cytoplasm around the nucleus, in blue, is observed. In the samples treated with compounds 8 and 14, besides a decrease in the cell numbers due to the antiproliferative effect, a disruption of the microtubule network can be observed in the three cell lines. After treatment with compounds 8 and 14, the HeLa and MCF7 cells acquired a rounded shape, whereas the U87MG cells lost their typical trigonal shape and adopted broader polygonal shapes. In the three cases, no microtubule fibers except for a green diffuse mass with no defined structure was observed in the treated cells.
This loss of microtubules and the cell morphology changes are in good agreement with the compounds affecting tubulin. Compound 9 caused similar effects in the microtubule in the MCF7 and HeLa cells. It also caused a microtubule network disruption in the U87MG cells, but to a lesser extent than compounds 8 and 14 since some tubulin fibers remained, and the cell shapes were not affected. The MCF7 cells treated with compounds 8 and 14 exhibited multilobulated nuclei, thus supporting the argument that the mitotic arrest caused by the impairment of the microtubule assembly is the mechanism at play [57].

Effects on the Cell Cycle
The effect of the selected compounds 8, 9, and 14 on the cell cycle distribution in the HeLa, MCF7, and U87MG cancer cell lines was studied via flow cytometry at different time points after the treatments (24, 48, and 72 h). The time-course analyses of the cell cycle showed different evolution depending on the compound and the cell line.
The non-treated HeLa cells (negative controls) showed similar cell cycle profiles regardless of the time point, with most of the cell population in the G0/G1 phases (50-69%), 24-37% of cells in the G2/M, and only 0.4-1.1% of cells at the subGo/G1 region (Figure 4). High percentages of cells were arrested at G2/M (80, 56, and 85% for compounds 8, 14, and 9, respectively) 24 h after the treatments. Besides these differences, 48 h after incubation, the mitotic arrest was similar for the three compounds (58, 60, and 72% for compounds 8, 14, and 9, respectively), and the subG0/G1 populations reached 14-18%. At a later time point of 72 h, the G2/M population decreased at the expense of an accumulation of sub G0/G1 fractions, which is indicative of cell death; this consisted of 52, 43, and 39% of cells in the subG0/G1 region for compounds 8, 14, and 9, respectively. This reveals that the antiproliferative effect of the three compounds on the HeLa cells is due to a mitotic arrest that begins only 24 h after the treatment and is followed by cell death induction.
The cell cycle profiles of the untreated MCF7 cells (negative controls) at 24, 48, and 72 h were similar to the HeLa profiles ( Figure 5). However, the effects of the selected compounds on this cell line were different than on the HeLa cells. The three compounds triggered mitotic arrest 24 h after the treatments (32, 40, and 56% for compounds 8, 14, and 9, respectively). Compound 9 was able to maintain the cell populations at G2/M, but the subG0/G1 fraction only reached 11% 72 h after the treatment, thus indicating that compound 9 disrupts mitosis in the MCF7 cells, but the cell death induction is not so strong. The profiles for compounds 8 and 14 evolved differently. The mitotic arrest caused 24 h after the treatment by compound 14, the most potent against the MCF7 cells, slowly declined at 48 and 72 h (30-35%), accompanied by an accumulation of 27% of the cells at subG0/G1 48 h after incubation that increased to 33% at the final time point of 72 h. The mitotic arrest caused by compound 8 had a slow start as well as the cell death induction; 48 h and 72 h after incubation, the subGo/G1 population changed from 9 to 26%. The MCF7 cells are deficient in caspase-3, which can explain the difficulties of the compounds in inducing cell death compared to the HeLa cells [62].
The untreated U87MG cells displayed similar cell cycle histograms to those of the HeLa and MCF7 cells at 24, 48, and 72 h ( Figure 6). The most potent compound 8 caused a severe mitotic arrest (39% in G2/M) and cell death induction (41% in subG0/G1) only 24 h after the treatment, followed by a progressive increase in the latter at the expense of the former over time, with the combined populations staying roughly constant at 85%. Compound 14 showed a maintained G2/M arrest and cell death induction at the three time points, staying globally at roughly 75%. Compound 9, after a slower start with a global G2/M (41%) plus subG0/G1 (17%) of 58%, also stabilized at later time points at 75%, again with the increased contribution of the apoptotic subG0/G1 population (46% at 48 h and 51% at 72 h) at the expense of the G2/M population (30% at 48 h and 26% at 72 h) observed for the other two compounds. This could explain the immunofluorescence results (observed 24 h after the treatments), where the U87MG cells did not appear to be too affected by compound 9 in comparison with the cells treated with compounds 8 and 14.  The cell cycle profiles of the untreated MCF7 cells (negative controls) at 24, 48, and 72 h were similar to the HeLa profiles ( Figure 5). However, the effects of the selected compounds on this cell line were different than on the HeLa cells. The three compounds triggered mitotic arrest 24 h after the treatments (32, 40, and 56% for compounds 8, 14, and 9, respectively). Compound 9 was able to maintain the cell populations at G2/M, but the subG0/G1 fraction only reached 11% 72 h after the treatment, thus indicating that compound 9 disrupts mitosis in the MCF7 cells, but the cell death induction is not so strong. The profiles for compounds 8 and 14 evolved differently. The mitotic arrest caused 24 h after the treatment by compound 14, the most potent against the MCF7 cells, slowly declined at 48 and 72 h (30-35%), accompanied by an accumulation of 27% of the cells at subG0/G1 48 h after incubation that increased to 33% at the final time point of 72 h. The mitotic arrest caused by compound 8 had a slow start as well as the cell death induction; 48 h and 72 h after incubation, the subGo/G1 population changed from 9 to 26%. The MCF7 cells are deficient in caspase-3, which can explain the difficulties of the compounds in inducing cell death compared to the HeLa cells [62]. In summary, although different cell cycle profiles were registered, the three compounds, 8, 9, and 14, were able to arrest the cell cycle at G2/M in the HeLa, MCF7, and U87MG cells 24 h after the treatments. Then, a certain level of mitotic arrest was maintained for 48 and 72 h while the SubG0/G1 population gradually increased until it reached maximum values at the 72 h time point, which is in good agreement with the antiproliferative IC 50 measurements.

Cell Death Mechanistic Studies
To elucidate the cell death mechanism suggested by the accumulation in the subGo/G1 region 72 h after the treatments with compounds 8, 9, and 14, we studied the mechanism of cell death via dual channel flow cytometry experiments using double staining by fluorescein isothiocyanate-labeled Annexin V (AnV) and propidium iodide (PI). The HeLa, MCF7, or U87MG cells were incubated with compounds 8, 9, or 14 for 72 h, stained with AnV and PI, and analyzed via flow cytometry (Figure 7). The untreated cells were used as negative controls. The apoptotic cells are Annexin V positive due to the translocation of phosphatidylserine from the inner leaflet to the surface of the plasmatic membrane, whereas necrosis causes membrane disintegration, leading to PI permeation. The response of the cells to both stains allow us to determine if they are viable (PI−, AnV−), early apoptotic (PI−, AnV+), late apoptotic, secondary necrotic (PI+, AnV+), or only necrotic The untreated U87MG cells displayed similar cell cycle histograms to those of the HeLa and MCF7 cells at 24, 48, and 72 h ( Figure 6). The most potent compound 8 caused a severe mitotic arrest (39% in G2/M) and cell death induction (41% in subG0/G1) only 24 h after the treatment, followed by a progressive increase in the latter at the expense of the former over time, with the combined populations staying roughly constant at 85%. Compound 14 showed a maintained G2/M arrest and cell death induction at the three time points, staying globally at roughly 75%. Compound 9, after a slower start with a global G2/M (41%) plus subG0/G1 (17%) of 58%, also stabilized at later time points at 75%, again with the increased contribution of the apoptotic subG0/G1 population (46% at 48 h and 51% at 72 h) at the expense of the G2/M population (30% at 48 h and 26% at 72 h) observed for the other two compounds. This could explain the immunofluorescence results (observed 24 h after the treatments), where the U87MG cells did not appear to be too affected by compound 9 in comparison with the cells treated with compounds 8 and 14. In agreement with the cell cycle results, most of the treated HeLa cells suffered from apoptotic induction 72 h after the treatments, and the three compounds had similar behaviors. A total of 93% of the HeLa cells treated with the most potent compound 8 underwent early and late apoptosis, whereas nearly 75% of apoptosis was observed for the less potent compound 9, and 86% of apoptotic cells were caused by compound 14 (Figure 7).
The percentages of the early and late apoptotic cells were higher than the fractions of the cells in the subG0/G1 phase, which can be explained by the different techniques employed in each assay. For the cell cycle experiments, a single PI staining after permeabilization is used. This means that many of the cells whose DNA content corresponds to the G2/M or even G0/G1 phases are probably beginning the phosphatidylserine translocation, but since their DNA is not yet affected, PI staining does not discriminate apoptotic cells. When double PI/AnV staining is performed, the apoptotic cells that were included in the G2/M or G1/G0 fractions in the cell cycle analysis respond positively not only to PI, but also to Annexin V. In summary, although different cell cycle profiles were registered, the three compounds, 8, 9, and 14, were able to arrest the cell cycle at G2/M in the HeLa, MCF7, and U87MG cells 24 h after the treatments. Then, a certain level of mitotic arrest was maintained for 48 and 72 h while the SubG0/G1 population gradually increased until it reached maximum values at the 72 h time point, which is in good agreement with the antiproliferative IC50 measurements.

Cell Death Mechanistic Studies
To elucidate the cell death mechanism suggested by the accumulation in the subGo/G1 region 72 h after the treatments with compounds 8, 9, and 14, we studied the mechanism of cell death via dual channel flow cytometry experiments using double staining by fluorescein isothiocyanate-labeled Annexin V (AnV) and propidium iodide (PI). The HeLa, MCF7, or U87MG cells were incubated with compounds 8, 9, or 14 for 72 h, stained with AnV and PI, and analyzed via flow cytometry (Figure 7). The untreated cells were used as negative controls. The apoptotic cells are Annexin V positive due to the translocation of phosphatidylserine from the inner leaflet to the surface of the plasmatic membrane, whereas necrosis causes membrane disintegration, leading to PI permeation. The response of the cells to both stains allow us to determine if they are viable (PI−, AnV−), early apoptotic (PI−, AnV+), late apoptotic, secondary necrotic (PI+, AnV+), or only Regarding the MCF7 cells (Figure 7), only compound 14 was able to induce apoptosis to a similar degree as observed for the HeLa cells (71%). Compound 8 also caused cell apoptosis, but to a lesser extent (47%), showing 46% of alive cells, and compound 9 displayed a very similar profile to the control cells since only 12% of cells underwent apoptosis. These results are consistent with the cell cycle profiles whose analyses revealed that mitotic arrest in MCF7 is not always followed by cell death. This behavior of the MCF7 cells can be attributed to their deficiency in caspase-3, which plays an essential role in the signal cascade of apoptosis.
Higher levels of apoptosis were registered for the treated U87MG cells compared with the MCF7 cells, but for the three treatments, 12-16% of cells were still alive at 72 h. As expected, a strong apoptotic response was observed after the treatment with the most potent compound 8, showing most cells in early or late apoptosis (80%). A lower response to Annexin V was displayed by the cells treated with compounds 9 and 14 (71% and 67%, respectively), showing similar profiles, which is in agreement with their similar antiproliferative potencies.
For the HeLa and MCF7 cells, the percentage of alive cells after 72 h of treatment correlates with the antiproliferative IC 50 values. On the other hand, the percentage of alive U87MG cells remains at a roughly constant value irrespective of the potency of the compounds.  In agreement with the cell cycle results, most of the treated HeLa cells suffered from apoptotic induction 72 h after the treatments, and the three compounds had similar behaviors. A total of 93% of the HeLa cells treated with the most potent compound 8 underwent early and late apoptosis, whereas nearly 75% of apoptosis was observed for the less potent compound 9, and 86% of apoptotic cells were caused by compound 14 (Figure 7).
The percentages of the early and late apoptotic cells were higher than the fractions of the cells in the subG0/G1 phase, which can be explained by the different techniques employed in each assay. For the cell cycle experiments, a single PI staining after permeabilization is used. This means that many of the cells whose DNA content corresponds to the G2/M or even G0/G1 phases are probably beginning the phosphatidylserine translocation, but since their DNA is not yet affected, PI staining does not discriminate apoptotic cells. When double PI/AnV staining is performed, the apoptotic cells that were included in the G2/M or G1/G0 fractions in the cell cycle analysis respond positively not only to PI, but also to Annexin V.
Regarding the MCF7 cells (Figure 7), only compound 14 was able to induce apoptosis to a similar degree as observed for the HeLa cells (71%). Compound 8 also caused cell

Lactate Dehydrogenase Assays
An analysis of treated cells via flow cytometry after simple (PI) or double (PI/AnV) staining, together with the immunofluorescence experiments, revealed that the antiproliferative effects of the selected compounds through MTT assays are explained by the induction of mitotic arrest, which occurs 24 h after incubation, which leads to apoptotic cell death 72 h later. All these experiments are complementary and accounted for different aspects of the cell damage caused by the compounds. The LDH leakage assay measures the release of lactate dehydrogenase (LDH) into the culture medium after cell membrane damage. It is a very interesting experiment because it can detect low levels of cell membrane damage that are not detected by other methodologies. The selected compounds 8, 14, and 9 showed to be particularly potent against glioblastoma cell lines. This is an important issue, considering that the only approved drug for glioblastoma treatment, temozolomide, has a very low antitumoral effect and is associated with resistance mechanisms.
To further study the effects caused by the compounds in glioblastoma cells and their capacity to discriminate between tumor and healthy cells, LDH experiments were carried out in both the glioblastoma U87MG cells and non-tumorigenic HEK-293 cells. The U87MG cells were treated for 72 h with concentrations of compounds from 1 nM to 1 µM, whereas the HEK-293 cells were incubated at higher concentrations, from 0.1 µM to 1 mM. These experiments revealed that the selected compounds caused further cellular damage than what was inferred by previous experiments, even at low nanomolar concentrations ( Figure 8). An amount of 1 nM of compound 8 caused nearly 40% of LDH release in the U87MG cells, which was increased to over 60% at 10 nM and reached 100% at 100 nM, meaning that total cell death is achieved at that concentration. Compounds 9 and 14 also provoked 100% of cell death above 100 nM. Although the antiproliferative IC 50 values of compounds 9 and 14 were in the double-digit nanomolar range, considerable amounts of released LDH, around 10% and 20%, were registered at concentrations as low as 1 and 10 nM, respectively, for both compounds, thus demonstrating a strong capacity to damage tumor cells. The LDH release values are consistent with the antiproliferative values obtained using the MTT method. Consistent with the antiproliferative assays, the HEK-293 cells were less sensitive to the selected compounds than the U87MG cells. No significant cytotoxic effects were observed for the HEK-293 cells 72 h after the treatment with compound 14 in a concentration range between 0.1 and 1 µM, whereas the three compounds displayed roughly 10% of LDH release at 10 µM. It is important to note that the three compounds reached their maximum cytotoxic effect in the U87MG cells at 100 nM and, for that concentration, no significant cell damage was detected by the LDH assays. At a higher concentration of 100 µM, the LDH release was only 20% for compound 9, and nearly 30% for compounds 8 and 14. Interestingly, the maximum levels of cytotoxicity were not above 40% at concentrations as high as 1 mM. These results confirm the high selectivity of the compounds, with the ability to cause a higher cytotoxic effect in tumor cells compared with healthy cells.

Structural Effects of the Bridge Modifications
The DFT calculations show that the amines preferentially adopt an extended conformation. On the other hand, the preferred dispositions for the acylated analogs correspond to a folded arrangement that places the aromatic moieties in a close, non-planar disposition similar to that observed for Z-stilbenes, such as the active configuration of combretastatin A-4 and most ligands occupying sites A and B of the colchicine site [63]. These dispositions are those found as the preferred docked poses (see later), thus suggesting that it has an important role in the different observed binding modes. Furthermore, the pre-arrangement of the free ligands in the bound disposition should favor binding and Consistent with the antiproliferative assays, the HEK-293 cells were less sensitive to the selected compounds than the U87MG cells. No significant cytotoxic effects were observed for the HEK-293 cells 72 h after the treatment with compound 14 in a concentration range between 0.1 and 1 µM, whereas the three compounds displayed roughly 10% of LDH release at 10 µM. It is important to note that the three compounds reached their maximum cytotoxic effect in the U87MG cells at 100 nM and, for that concentration, no significant cell damage was detected by the LDH assays. At a higher concentration of 100 µM, the LDH release was only 20% for compound 9, and nearly 30% for compounds 8 and 14. Interestingly, the maximum levels of cytotoxicity were not above 40% at concentrations as high as 1 mM. These results confirm the high selectivity of the compounds, with the ability to cause a higher cytotoxic effect in tumor cells compared with healthy cells.

Structural Effects of the Bridge Modifications
The DFT calculations show that the amines preferentially adopt an extended conformation. On the other hand, the preferred dispositions for the acylated analogs correspond to a folded arrangement that places the aromatic moieties in a close, non-planar disposition similar to that observed for Z-stilbenes, such as the active configuration of combretastatin A-4 and most ligands occupying sites A and B of the colchicine site [63]. These dispositions are those found as the preferred docked poses (see later), thus suggesting that it has an important role in the different observed binding modes. Furthermore, the pre-arrangement of the free ligands in the bound disposition should favor binding and result in a potency increase.

Docking Studies
The binding modes of the synthesized compounds (Figure 9) at the colchicine site of the tubulin were studied via ensemble docking experiments. X-ray crystal structures of the colchicine site ligands bound to the tubulin have shown that it can bind compounds of varied sizes and nature, more than what was initially thought, occupying different subpockets even for structurally related ligands [63]. Therefore, we accounted for the colchicine site flexibility by using in the docking experiments an ensemble of X-ray structures of tubulin in complexes with a set of diverse colchicine site ligands that represent different protein arrangements and ligand pharmacophore configurations, and therefore, efficiently explored the possible interaction space in the docking experiments [64]. We selected 60 X-ray crystal structures [65], plus 6 additional ones from a previous molecular dynamics simulation [66] obtained as described, to represent the different possible configurations of the colchicine site of the tubulin. of varied sizes and nature, more than what was initially thought, occupying different subpockets even for structurally related ligands [63]. Therefore, we accounted for the colchicine site flexibility by using in the docking experiments an ensemble of X-ray structures of tubulin in complexes with a set of diverse colchicine site ligands that represent different protein arrangements and ligand pharmacophore configurations, and therefore, efficiently explored the possible interaction space in the docking experiments [64]. We selected 60 X-ray crystal structures [65], plus 6 additional ones from a previous molecular dynamics simulation [66] obtained as described, to represent the different possible configurations of the colchicine site of the tubulin. Two docking programs with very different scoring functions (PLANTS [67] and Au-toDock 4.2 [68]) were used for the docking experiments. For every docked ligand, we took the binding pose as the common pose that was the best scoring combination for the two docking programs as assessed via automated geometry and scoring comparison procedures. The geometry assessment consisted of the following: (a) sub-pocket occupancy assignments for each ligand pose calculated by measuring the lowest distances to the subpocket geometrical centers as determined by the pharmacophores derived from the X-ray Two docking programs with very different scoring functions (PLANTS [67] and AutoDock 4.2 [68]) were used for the docking experiments. For every docked ligand, we took the binding pose as the common pose that was the best scoring combination for the two docking programs as assessed via automated geometry and scoring comparison procedures. The geometry assessment consisted of the following: (a) sub-pocket occupancy assignments for each ligand pose calculated by measuring the lowest distances to the subpocket geometrical centers as determined by the pharmacophores derived from the X-ray structures of the colchicine site ligands in complex with tubulin; (b) RMSD calculations between the poses and unsubstituted scaffolds with the same bridges that place the phenyl rings at the centers of the A, B, or C sites of the colchicine site of the tubulin; and (c) the RMSDs of the poses with those of the pdb [69] X-ray structures of combretastatin A-4 (for A-B binding), MI-181 (for A-C binding), or ABT-751 (for A-B-C binding) (pdb IDs 5LYJ, 4YJ2, and 3HKC, respectively). Accordingly, every pose for every ligand is allocated to the corresponding sub-pockets. To make the inter program scores comparable, the individual scores were converted to relative scales where they rank between zero (worse) and one (best) and to Z-scores. Subsequently, we assigned a consensus binding mode for each ligand to the pair of poses common to the two programs with the best possible Z-values for each. The control experiments with representative colchicine site ligands of the known X-ray structures in complex with tubulin correctly retrieved the same experimental poses as before [16].
Amines 1 to 6 bind at the A (placed above sheets S9, S8, and S10 along the A to C direction and below helix H8) and C (placed with sheets S5, S6, and S7 below and helix H7 above) zones of the colchicine site of the tubulin ( Figure 9A) [17]. The methoxy benzothiazole amines bind with the methoxy phenyl ring deepest in the β subunit occupying a hydrophobic pocket surrounded by the sidechains of Phe20β (H1), Phe169β (S5), Met235β (H7), the thiazole nitrogen, and the amine contacting Glu200β (S6) and Tyr202β (S6) at the C zone, and with the other aromatic rings occupying the A pocket, above the sidechains of Ile378β (S10), Ala316β (S8), and Ile318β (S8) and behind Leu255β (H8).
The acylation of the methoxy benzothiazole amines 1-6 to formamides 13-18, acetamides 19-24, and ethylureas 7-12 results in an alternative binding mode at the B and A zones ( Figure 9C,D). This change is due to the steric hindrance of the acyl moieties and the induction of a preference for a "folded" arrangement of the two aryl systems, a disposition required for the binding to the A-B zone, and a well-documented requirement for related systems. In these series, the methoxy-benzothiazole occupies the B zone, stacked behind helix H8 and with the sidechain of Asn258β (H8) piled above the phenyl ring that makes hydrophobic contacts with Met259β (H8), Thr314β (S8), and the methylenes of Lys352β (S9). The acyl moieties protrude toward the interfacial space between the subunits, and the other aromatic moieties occupy the A zone, filling from above the pocket formed by the sidechains of Ala316β (S8), Ile318β (S9), Lys352β (S9), and Ala354β (S9), capped by Ala250β (H7), Leu255β (H8), and with the pyridine ring orienting its nitrogen atoms toward the thiol group of Cys241β (H7). The optimal arrangement of the pyridine nitrogen is observed for the 3-pyridyl analogs, which is in good agreement with the biological results.
The ensemble docking approach used not only selects the most favorable poses for each ligand, but also provides information about the preferred protein structures that give these poses. In this respect, the amine derivatives 1-6 binding at zones A-C are selected for the protein structures that have ligands in these same sub-pockets. The most frequent pdb ID retrieved is 5YL4, whose ligand is a benzophenone analog of plinabulin ( Figure S25 in the Supplementary Material) that deeply inserts the benzophenone moiety in the subunit. For the acyl derivatives binding within the A and B zones, the most retrieved structures are 5H7O, with a 2-(1H-indol-4-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo [4,5-c]pyridine ligand; 5JVD, with a (2E)-3-(3-hydroxy-4-methoxyphenyl)-1-(7-methoxy-2H-1,3-benzodioxol-5-yl)-2-methylprop-2-en-1-one; and 5LYJ, the complex of tubulin with combretastatin A-4 ( Figure S25 in the Supplementary Material). These three have a trimetho-xyphenyl ring or related moiety within the A pocket. Additionally, as previously observed, the proteins in every pair of consensus poses are different for AutoDock and PLANTS, which probably reflects that the different scoring functions of both programs select different protein configurations. These results validate the use of as many protein structures as possible in the docking experiments (ensemble docking strategy) and reinforce the application of consensus scoring approaches. In summary, the docking studies support the argument that ligands can bind to the colchicine site on the tubulin, targeting zones A-C for amines 1-6 and zones A-B for the rest of the amide derivatives. The most favorable interactions were found for the pyridine derivatives with ethylurea and formamide functionalities, thus explaining the antimitotic activity and the structure-activity relationships observed for each series of compounds.

Conclusions
One of the main limitations of the colchicine site ligands is their low aqueous solubility due to the hydrophobic nature of the binding site. We succeeded in synthesizing a new series of water-soluble benzothiazole derivatives that are able to target the colchicine binding site, inhibit tubulin polymerization, and behave as antiproliferative agents in different kinds of cancer cells. The replacement of the olefin linker of MI-181 or CA-4 by methylamine, acetamide, formamide, or ethylurea functionalities was proven to significantly increase, in most cases, the polar surface area and the water solubilities of the synthesized 6-methoxy derivatives with respect to the reference compounds. The structure-activity relationship studies revealed that the combination of 6-methoxibenzothiazole with a pyridine ring provides an optimal potency as antiproliferative agents and tubulin polymerization inhibitors, particularly when combined with ethylurea or formamide groups. Whereas the pyridine derivatives with amino groups (1-6) showed antiproliferative IC 50 values in the micromolar range, the best performing compounds (pyridine-based formamide and ethylurea derivatives) reached nanomolar values for some cell lines, showing an especially potent antiproliferative profile against the difficult-to-treat glioblastoma cell lines. The studies on the mechanism of action of the most potent compounds revealed that compounds 8, 9, and 14 disrupt the microtubule network of treated cancer cells and arrest the cell cycle at the G2/M phase 24 h after the treatment, followed by the induction of apoptotic cell death. The compounds exhibited a high selectivity toward cancer cells with respect to the non-tumoral HEK-293 cells and were not susceptible to multidrug resistance efflux pumps such as MDR1/P-gp. Binding at the colchicine site is supported by computational studies that suggest highly favorable interactions at the A-B zones for the most biologically active compounds and provide a structural explanation for the structure-activity relationships. The ensemble of results sustains the success of the strategy of design and provides new possibilities to discover synthetically accessible anticancer drugs, targeting the colchicine site of the tubulin, with improved water solubility. The acylation of the amino group of the benzothiazole derivatives was favorable in terms of water solubility and biological activity, so the incorporation of new polar functionalities on the amino group could lead to new active colchicine site ligands. The pharmacodynamic and pharmacokinetic properties of the obtained compounds show a high potential for further development. According to the high potency, aqueous solubility, and selecti-vity against glioblastoma cell lines of compounds 8 and 14, they are good candidates to be used in a glioblastoma mouse orthotopic xenograft model to study their capacity to reduce the volume of the tumor. This could open new doors in the development of novel chemotherapy agents for the treatment of glioblastoma.